Band 3 is the predominant polypeptide of the human erythrocyte membrane. We propose a systematic study of the relationship of the structure of this polypeptide to its integration and function within this membrane. Preliminary data indicate that component 3 behaves as a hydrophobic, dimeric glycoprotein, comprising roughly 25 percent of the membrane protein maps. It appears to span the thickness of the membrane asymmetrically, with its carbohydrate groups confined to the external surface and binding sites at its inner surface for cytoplasmic enzymes. We have solubilized and purified this protein (free of lipid) in a non-denaturing detergent system and its amino acid composition has been established. It is our aim to elucidate the primary structure of the band 3 polypeptide chain in the membrane, using the split products derived from limited selective proteolysis in situ together with fragments generated by chemical cleavage of the topagraphically-labeled molecule. Using impermeable ghosts and inside-out vesicles, each membrane face will be selectively reacted with covalent radioactive tags or proteases. The regions unique to each surface, as well as inaccessible core segments, will be characterized with respect to the parent chain terminal, sugar and amino acid content and amino acid sequence. Ultimately we will attempt to relate these structural features to the apparent functional role of band 3 as a transport site for anions and perhaps other solutes.